Preparation and Characterization of Composite Ceramic Nanoparticles (ZnO-Ag) via Green Synthesis for Medical Applications

Recent years have witnessed a notable rise in the "green synthesis" of silver (Ag)/zinc oxide (ZnO) nanocomposites using plant-based products and biological organisms, a method valued for its eco-friendliness and the resulting potent antibacterial properties. Recent studies indicate that these nanocomposites exhibit superior performance compared to ZnO or Ag alone [1-3]. The interested in applying biological materials and their effects within the human body, preceded by many analyzes and tests that confirmed their effectiveness. Including metals (titanium alloys and others), ceramics (hydroxyapatite) and polymers of all kinds. Research has varied and varied in the manufacturing, coating, or mixing mechanism to arrive at materials with sound effectiveness, biocompatibility, and low risk within the human body [4-7]. Recently, materials have appeared that have proven their chemical and biological effectiveness in the body, and among these materials are Ag and ZnO. The methods for preparing these materials tend toward manufacturing nanomaterials by physical, chemical, and biological means, and each method has its own distinct characteristics in terms of shape and effects. To ensure environmental and in vivo safety, a biosynthetic approach, especially plant-derived synthesis, is the optimal strategy. Nano-sized Ag and ZnO particles are highly effective as antimicrobial, drug delivery, and anti-cancer cell systems [5]. Nanomaterials and their effective applications in many biomedical and healthcare applications, including ZnO nanoparticles (NPs), have emerged due to their high biocompatibility, low toxicity, cost and excellent biocompatibility, which has facilitated their use as a practical alternative to hazardous traditional raw materials. They are effective against a variety of respiratory viruses, including SARS-CoV-2, and have shown promising results in preventing, diagnosing, and treating COVID-19. They are also used in other applications such as supplements, food additives, and semiconductor materials due to its low toxicity. ZnO is an inert material. When exposed to light, it absorbs energy and turns into a semiconductor, which leads to the release of zinc ions. Molecular effects resulting from the semiconducting or electronic properties of ZnO NPs produce reactive oxygen species (ROS) as a result of the release of excess zinc ions. To reduce the toxicity of ZnO NPs, their surface is coated with silica, which prevents the release of zinc ions (Zn²⁺) and the generation of ROS. This reduces their toxicity and allows them to be used as a biological and bactericidal agent [4-8]. Ag, especially its NPs, is an effective antibacterial agent, and many methods have been developed to prepare it free of toxic chemical components. We prepared Ag NPs using a biological method, where plant extracts of Artocarpus heterophylus and Azadirachta indica were used as reducing and stabilizing agents in the process of synthesizing Ag NPs. Another study used an environmentally friendly method using plant extracts such as flowers, leaves, bark, roots, and stems as antimicrobials, antifungals, and anti-cancer agents, using whole plant parts [9-11]. Ag/ZnO NPs with a size of 30-50 nm were prepared using an environmentally friendly plant extract method, where Amla (Phyllanthus emblica) seed extract was used as a reducing agent, and this method showed antibacterial activity. The antibacterial activity against Salinivbrio proteolyticus, Bacillus subtilis, and Staphylococcus epidermidis, dl has been evaluated, and the compound can be used in medical applications such as wound healing and medical dressings [12]. Other research has also been conducted to develop Ag-ZnO NPs in an environmentally friendly manner. Eclipta prostrate (EP), Eclipta alba (EA). A multi-herbal extract was used to manufacture Ag-ZnO nanocomposites (Ag-ZnO NCs) via the sol-gel method at different concentrations; the Ag-ZnO NPs, at a concentration of 50 μg, achieved higher antimicrobial activity than the extract alone and high chemical stability for 90 days [13, 14]. In another study, four metal NPs (NPs) with propolis extract (Ext) with NPs including ZnO/Ext, ZnO/Ag/Ext, ZnO/CuO/Ext, ZnO/Ag/CuO/Ext, and ZnO/W, were prepared by microwave method; Fourier transform infrared (FTIR) spectroscopy, TGA, and Brunauer-Emmett-Teller analyses. The results have proven their importance in direct application to tissues (inside the body), accelerating wound healing [15, 16]. The literature supports principally three mechanisms that have been observed together or separately, by which AgNPs exert their antibacterial action [17-19]. The first one postulates that AgNPs act at a membrane level, as they can penetrate the outer membrane, accumulating in the inner membrane where the adhesion of the NPs to the cell generates their destabilization and damage, increasing membrane permeability and inducing leakage of cellular content and subsequently its death [20, 21].

3.1 Manufacturing of nano-silvar and ZnO by chemical method

NPs were prepared using the green method with a plant extract from Laurus nobilis leaves.

Dried leaves of Laurus nobilis were collected, ground, and 5 g were dissolved in 100 mL of deionized water. The solution was then placed on a magnetic stirrer (IKA C-MAG HS 7, German) for 5 hours at 60 ℃. Afterward, it was filtered by centrifugation to remove all impurities. The phytochemicals present in Laurus nobilis leaf extract, such as polyphenols and flavonoids, act as natural reducing and capping agents by reducing metal ions, promoting nanoparticle nucleation and growth, and stabilizing the synthesized NPs.

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Figure 1. Method of preparation of (Ag, ZnO, ZnO₇₅-Ag₂₅ and ZnO₅₀-Ag₅₀) nanopowder

•Zinc acetate concentration was approximately 0.24 M by dissolving 4.2 g of zinc acetate salt in 80 mL of deionized water, placed on a magnetic stirrer, and adding 20 mL of the plant extract at 60 ℃ for 3 hours. A colour change in the solution was observed, indicating the formation of a suspended nanomaterial. The solution was then filtered using filter paper and washed three times with deionized water and ethanol. Finally, it was dried and calcined at 450 ℃ for 3 hours to obtain ZnO NPs, with an alkaline medium (pH = 11).

•Ag is also prepared by dissolving approximately 0.074 M 1.0 g of Ag nitrate (AgNO₃) in 80 mL of deionized water and adding 20 mL of plant extract drop by drop at 60 ℃, stirring with a magnetic stirrer for 3 hours. Afterward, it is filtered using a centrifuge at 9000 rpm, washed with water and alcohol, dried at room temperature, and calcined at 450 ℃ for 3 hours to obtain nano-Ag in an alkaline medium (pH = 10).

•Two composite solutions were mixed with volume ratios of 75:25 and 50:50. were  prepared (75 ZnO+ 25 Ag) and (50 ZnO + 50 Ag), each composite placed on a magnetic stirrer at 60 ℃ for 3 hours. The solution is separated by centrifugation, washed with water and alcohol, dried at room temperature, and calcined at 450 ℃ for 3 hours; pH = 10-11.

•Four samples were prepared (Ag, ZnO, ZnO₇₅-Ag_25, ZnO₅₀-Ag₅₀), as shown in Figure 1.

3.2 Bacterial preparation

Mueller Hinton broth and agar (HIMEDIA, India). After autoclaving for 15 minutes at 121 ℃ and 15 pounds per square inch of pressure, the medium cooled to 45 ℃ before being poured into Petri dishes. The disk diffusion technique was utilized to ascertain the aqueous extract's sensitivities in vitro. Using the direct colony suspension method, Petri plates containing Mueller-Hinton agar were inoculated to a density of 1 × 10⁸ bacterial cells/ml in Mueller-Hinton broth (MHB), or 0.5 McFarland standard. Following a wipe onto the plates, 100 µl of the activated microbe solution was allowed to sit at room temperature for ten minutes. Utilizing a sterile stainless steel cork borer, four wells with identical dimensions and depth (6 mm in diameter) were created in the agar. Different concentrations of the extract (20 mg/ml, 40 mg/ml, and 60 mg/ml) were added to each well, while chlorhexidine digluconate (CHX) (0.2%) was added to the fourth well as a positive control. The plates underwent an aerobic 18-24-hour incubation period at 37 ℃ [22].

5.1 X-ray diffraction test

 The structural properties of the samples were analysed using XRD. Figure 2 showed the formation of distinct Polycrystalline nanomaterial phases for both ZnO and Ag, ZnO₇₅ + Ag₂₅ and ZnO₇₅ + Ag₂₅. For the ZnO sample, the pattern showed distinctive diffraction peaks belonging to the hexagonal structure, for 2θ⸰ (32, 34, 36, 57, 63, 68, 77) corresponding to different crystallographic planes (100), (002), (101), 102), (110), (103), (112) with card Joint Committee on Powder Diffraction Standards (JCPDS) (01-075-1526) [23]. While as for the Ag 2θ⸰ (38, 44, 65, 77) respectively, corresponding miller indices (111), (200), (220), (311) which is consistent with the cubic structure of Ag [24], indicating the successful formation of Ag NPs is the known structure Which matches card JCPDS (00-004-0783). As for the sample prepared with different ratios of ZnO₇₅ + Ag₂₅, peaks of ZnO were observed, while Ag peaks appeared with less intensity, such as the peak at 2θ⸰ (38.1). This indicates the presence of Ag in the prepared sample without a change in the crystal structure. A slight shift was also observed due to interference or lattice stress. However, in the ZnO₅₀ + Ag₅₀ sample, the intensity of the Ag peaks increased significantly, especially at the2θ⸰ 38° and 44° angles, with the continued appearance of the ZnO peaks [25]. This indicates the simultaneous presence of two phases in the material. Furthermore, the widening of the peaks may indicate a small crystal size, which is consistent with [26]. The crystal size (D) and full width at half maximum (FWHM) were calculated using the Scherrer Eq. (1) [27]:

$D=\frac{0.9 \lambda}{\beta \cos \vartheta}$        (1)

where,

D: crystallite size measured in nanometers.

β: full Width at Half Maximum measured in radians.

λ: wavelength of X-ray measured (0. 15406 nm).

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Figure 2. X-ray diffraction (XRD) pattern comparison for ZnO, Ag, ZnO₅₀ + Ag₅₀ and ZnO₂₅ + Ag₇₅

The results showed that the crystal diameter falls within the nanoscale range for ZnO and Ag; the composite ZnO₇₅ + Ag₂₅ and ZnO₅₀ + Ag₅₀ were 23.8, 52, 25, and 23 nm, respectively. A slight increase in crystal size was observed compared to ZnO. This is attributed to the interaction of Ag with ZnO, resulting in crystal growth due to the presence of Ag. The small particle size is also attributed to the presence of a plant extract, which acts as a reducing and coating agent, thus contributing to the small size of the materials prepared using the green method, as shown in Table 1. Regarding the XRD analysis results—specifically the peak broadening (increased FWHM) observed due to the green chemistry synthesis method—the widening of the peak indicates a reduction in particle size. This confirms the efficiency of the synthesis method for producing the nanomaterial, a finding consistent with the cited sources [28-31].

Table 1. The structural parameters of the ZnO, Ag, ZnO₅₀ + Ag_50, and ZnO₂₅ + Ag₇₅ samples

Table 2 shows the lattice constants (a, c) for the ZnO (Hexagonal Wurtzite) crystal structure; the values were calculated using Eqs. (2) and (3) (for ZnO, there are two unknowns in the equation (a and c); therefore, we choose the vertices so that the equation contains only one unknown. While for Ag (Face-Centered Cubic) there is only one unknown, the lattice constant (a) was calculated by using Eqs. (3) and (4). The ZnO-Ag composite doesn’t have a new crystal structure. Rather, it is a biphasic nanocomposite, and the lattice constant was also calculated using Eqs. (2) and (3).

$d=\frac{y}{2 \sin \theta}$        (2)

$\frac{1}{d^2}=\frac{4}{3}\left(\frac{h^2+h k+k^2}{a^2}\right)+\frac{l^2}{c^2}$        (3)

$a=d \sqrt{h^2+k^2+l^2}$        (4)

where,

(hkl): Miller indices.

d: Interplanar spacing.

Based on the literature review the lattice constant for ZnO are (a = 3.249, c = 5.206) A⸰ and for Ag the value of lattice (a = 4.086) A⸰. Observing the results in Table 2, we note the proximity of the values for the first ZnO and second Ag samples individually [32, 33]. There is no single standard value for the lattice constant of the ZnO/Ag composite, due to variations in crystal structure and preparation method. We observe that the value of the lattice constant (c)for the composite samples ZnO₇₅: Ag₂₅ and ZnO₅₀: Ag ₅₀ is close to its value in the single sample ZnO, while the value of the lattice constant (a) is closer to that of Ag than to that of ZnO. This behavior indicates that the Ag did not form a new crystalline phase with the ZnO; instead, it exists as a separate phase on the ZnO surface or at its grain boundaries (forming a heterojunction). Given that ZnO possesses a hexagonal (wurtzite) structure, the c-axis remains relatively stable and less affected by the presence of Ag particles, thus remaining close to its standard value. The variation in the a-axis parameter is attributed to lattice strain and interactions at the ZnO/Ag interface, resulting in a slight shift in lattice constants-particularly along the basal directions-while both ZnO and Ag retain their respective crystalline phases [34].

Table 2. The lattice constants of the ZnO, Ag, ZnO₅₀ + Ag_50, and ZnO₂₅ + Ag₇₅ samples

5.2 Energy-dispersive X-ray spectroscopy test

The EDX elemental analysis can be identified by peaks for ZnO, Ag, ZnO₅₀ + Ag_50, and ZnO₂₅ + Ag₇₅ NPs, as shown in Figures 3-6, close to the tables assisted with Figures 3-6. The energy transitions that represent Zn (Lα = 1.012, Kα = 8.046, and Kβ = 9.5 KeV), O (Kα = 0.5 KeV) and Ag (Mα = 0.30, Lα = 2.89, Lβ = 3.15 and Lγ = 3.35 KeV). The EDX elemental analysis show the major peaks for Zn at Lα = 1.012 and especially at Kα 8.046, the presence of peaks at high energies signifiesand increasing in intensity with increasing the concentration, a transition that is mean translation for K level. While the Ag peaks concentrate near L ≈ 3 the major peak Lβ = 3.15 and Lγ = 3.35 KeV, it is requires a higher excitation voltage [35, 36].

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Figure 3. Energy-dispersive X-ray spectroscopy (EDX) elemental analysis for ZnO sample

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Figure 4. Energy-dispersive X-ray spectroscopy (EDX) elemental analysis for Ag sample

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Figure 5. Energy-dispersive X-ray spectroscopy (EDX) elemental analysis for ZnO₅₀ + Ag₅₀ sample

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Figure 6. Energy-dispersive X-ray spectroscopy (EDX) elemental analysis for ZnO₇₅ + Ag₂₅ sample

5.3 Field emission scanning electron microscopy test

The FESEM test images with magnification (5 µm to 500 nm) for all samples in Figures 7(a-h) have a size of about 44.65 nm to 100.08 nm. At high magnification (500 nm), the bright Ag particle clustering shows non-uniform morphology of the particles, with spherical, circular structures and oval and some asymmetric shapes; ZnO has spherical, circular structures and self-assembled star-like structures. The agglomeration clear in composite samples ZnO₅₀-Ag₅₀ more compact structure with smaller particles. Increasing the ZnO concentration ZnO₇₅-Ag₂₅ resulted in High density with large clumps homogeneous particle distribution. The agglomeration is due to high surface energy NPs, specifically ZnO [37].

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Figure 7. Field emission scanning electron microscopy (FESEM) image samples at different magnifications: a and b: ZnO sample; c and d: Ag sample; e and f: ZnO₅₀ + Ag₅₀ sample; and g-h: ZnO₇₅ + Ag₂₅ sample

5.4 Fourier transform infrared test

FTIR spectroscopy from 400 up to 4000 cm⁻¹ Range in Figure 8 were use to investigated nanopowders pure ZnO, Ag, composite ZnO₅₀ + Ag₅₀ and ZnO₇₅ + Ag₂₅ samples shown in Figure 8. In the ZnO, composite ZnO₅₀ + Ag_50, and ZnO₇₅ + Ag₂₅ samples, the absorption bands in the range between 437 and 513 cm⁻¹ correspond to the vibrations of Zn-O stretching, proving it is ZnO. The absorption bands at 1450 cm⁻¹ correspond to the vibrations of C-O/Zn-OH Surface links. The absorption bands in the range between 1645 and1647 cm⁻¹ correspond to the vibrations of H-O-H bending, production of ROS, and absorbed water bending. The absorption bands in the range between 3349 and 3441 cm⁻¹ correspond to the vibrations of O-H stretching. In Ag, the absorption bands in the range 500 cm⁻¹ correspond to the Ag-O bond vibration. The absorption bands in the range 1645 cm⁻¹ correspond to Ag.

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Figure 8. Fourier transform infrared (FTIR) analysis: ZnO, Ag, ZnO₅₀ + Ag₅₀ and ZnO₇₅ + Ag₂₅ samples

Shift occurs at vertices (composite ZnO₅₀ + Ag₅₀ and ZnO₇₅ + Ag₂₅ samples) this belong to change the chemical environment and interaction between Ag+ZnO lead to formation composite. The vibrations and ROS production are present in the four samples [38, 39].

5.5 Ultraviolet test

Figures 9(a)-(c), UV spectroscopy was used to calculate the band gap energy (Eg) for the nanopowders pure ZnO, Ag, composite ZnO₅₀ + Ag_50, and ZnO₇₅ + Ag₂₅ samples using Tauc Eq. (5) [40].

$(\alpha h v)^n=\mathrm{A}(h v-E g)$         (5)

where,

$\alpha$: Absorption coefficient (calculated from absorbance).

$h v$: Photon energy.

Eg: Band gap energy (in electron volts, eV).

A: Proportionality constant.

n: An exponent determining the nature of the electronic transition.

The values of Eg (2.85, 2.5, 2.6, and 2.9 eV) for ZnO, Ag, ZnO₅₀ + Ag_50, and ZnO₇₅ + Ag₂₅ samples, respectively. ZnO have large Eg value compared to value of Ag that’s due to stabilizer of ZnO [41]. After composite between ZnO₅₀ + Ag₅₀ sample the value of Eg decreasing, and ZnO₇₅ + Ag₂₅ sample the value of Eg returns to raise due to increasing the concentration of ZnO. A small Eg means that matter becomes more able to utilize energy from the surroundings and Increased production of ROS that lead to increased antibacterial activity rather than direct evidence of the mechanism, a cording to the reference [42] the antibacterial activity of ZnO is primarily linked to the generation of ROS. It is known that Ag element have surface plasmon resonance (SPR) phenomena and the ZnO work increased ROS production of (O₂^-, H₂O₂ and OH) that is clear in the result of FTIR analysis Figure 8 [43, 44]. A reduction in the ZnO band gap below the standard value of 3.2-3.3 eV can occur due to crystalline defects, the incorporation of Ag, or intra-gap energy states (defect states) [45].

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Figure 9. Ultraviolet (UV) analysis: (a) ZnO, (b) Ag, (c) ZnO₅₀ + Ag₅₀ and ZnO₇₅ + Ag₂₅ samples

5.6 The disk-diffusion technique on agar

The antibacterial activity measured inhibition zone data are based on single measurements. The disk diffusion was employed to test Gram-negative and Gram-positive bacteria against Staphylococcus aureus-positive and Pseudomonas aeruginosa-negative bacteria, and the inhibition diameter was recorded at different concentrations (100%, 50%, 25%, and 12.5% mg/mL). Figures 10-13 and Table 3 show the results of antibacterial activity of Ag, ZnO, and composite ZnO₇₅-Ag₂₅ and ZnO₅₀-Ag₅₀ samples, respectively, and recorded the highest inhibition diameter at a high concentration of 100 mg/mL. The effective effect against Staphylococcus aureus bacteria showed 22 mm for ZnO₅₀-Ag_50, more than Ag 19 mm, ZnO 17 mm, and 17 mm for ZnO₇₅-Ag₂₅. Pseudomonas aeruginosa, ZnO recorded the highest inhibition diameter of 24 mm at a concentration of 100 mg/mL, more than Ag 22 mm, 20 mm for ZnO₅₀-Ag₅₀, and 19 for ZnO₇₅-Ag_25, respectively. The results regarding the bacterial impact revealed significant variations across the test conditions, depending on the type, composition, and concentration of the substance, as well as the specific bacterial strain used. Notably, the observed effects intensified with increasing substance concentration, with peak values recorded at 100 mg/mL, indicating that bacterial activity is dependent on the amount of substance available to interact with the bacterial cells. For Staphylococcus aureus, the ZnO₅₀-Ag₅₀ composite exhibited the largest inhibition zone 22 mm, compared to 19 mm for Ag and 17 mm for both ZnO and ZnO₇₅-Ag₂₅. This enhanced activity can be attributed to a synergistic effect between Ag and ZnO at the 50: 50 ratio; Ag contributes by interacting with bacterial cell components, while ZnO facilitates surface interactions and the generation of ROS. The reduced inhibition observed with ZnO₇₅-Ag₂₅ indicates that increasing the ZnO proportion does not necessarily enhance activity, demonstrating that the ratio of the composite's components influences the efficiency of the interaction with the cells. Regarding Pseudomonas aeruginosa, ZnO exhibited the highest inhibition (24 mm), whereas the values were 22 mm for Ag, 20 mm for ZnO₅₀-Ag₅₀, and 19 mm for ZnO₇₅-Ag₂₅. This indicates that the response to the nanocomposites varies depending on the bacterial species. Such variation may be linked to differences in cell envelope composition; Gram-negative bacteria possess an additional outer membrane that can influence the permeability of particles and active agents, as well as their interaction with the cell surface. Consequently, reduced inhibition observed with certain Ag/ZnO compositions does not imply lower overall antibacterial activity; rather, it reflects differences in the material's suitability and its interaction mechanism with the specific type of bacteria. The efficiency of bacterial inactivation depends on the composition and concentration of the hybrid (Ag/ZnO) composite and its Eg; incorporating Ag into ZnO modifies the composite's electronic properties. The ZnO component contributes to the generation of ROS—such as O₂⁻, H₂O₂, and OH—which can interact with bacterial cell walls and DNA, while the SPR effect of the Ag enhances the antibacterial activity [46, 47].

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Figure 10. Antibacterial activity of Ag nanoparticles (NPs) using disk diffusion at concentrations (100%, 50%, 25%, 12.5%) against: (a) Staphylococcus aureus bacteria, (b) Pseudomonas aeruginosa bacteria

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Figure 11. Antibacterial activity of ZnO nanoparticles (NPs) using disk diffusion at concentrations (100%, 50%, 25%, 12.5%) against: (a) Staphylococcus aureus bacteria, (b) Pseudomonas aeruginosa bacteria

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Figure 12. Antibacterial activity of ZnO₅₀ + Ag₅₀ nanoparticles (NPs) using disk diffusion at concentrations (100%, 50%, 25%, 12.5%) against: (a) Staphylococcus aureus bacteria, (b) Pseudomonas aeruginosa bacteria

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Figure 13. Antibacterial activity of ZnO₇₅ + Ag₂₅ nanoparticles (NPs) using disk diffusion at concentrations (100%, 50%, 25%, 12.5%) against: (a) Staphylococcus aureus bacteria, (b) Pseudomonas aeruginosa bacteria

Table 3. Antibacterial activity of Ag, ZnO, and composite ZnO₇₅-Ag₂₅, ZnO₅₀-Ag₅₀ samples using disk diffusion against Staphylococcus aureus bacteria and Pseudomonas aeruginosa bacteria